Novel therapeutic treatment of chronic obstructive pulmonary disease

ABSTRACT

The present invention provides a method of treating or preventing a chronic obstructive pulmonary disease in a subject, comprising administering to said subject an amount of an agent affective to inhibit apoptosis of the subject&#39;s lung cells and thus treat or prevent chronic obstructive pulmonary disease in the subject. The present invention provides for a method of diagnosing the disease. Also, the invention provides a method for identifying a compound effective to treat or prevent a chronic obstructive pulmonary disease, comprising (a) contacting lung cells from a subject having a chronic obstructive pulmonary disease with the compound and measuring the level of apoptosis of the lung cells in the presence of said compound, (b) measuring the level of apoptosis of the lung cells from the same subject in the absence of said compound, (c) comparing the level of apoptosis in step (a) with the level of apoptosis in step (b), wherein a higher level of apoptosis in step (b) indicate that the compound is effective to treat or prevent chronic obstructive pulmonary disease.

[0001] This application is a continuation-in-part of U.S. Ser. No.09/514,885, filed Feb. 29, 2000, the contents of which are herebyincorporated by reference.

[0002] Throughout this application, various publications are cited byreference numbers. Full citations for these publications may be foundlisted at the end of the specification immediately preceding the claims.Certain references and publications are cited by full citation. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art as known to those skilled therein as ofthe date of the invention described and claimed herein.

BACKGROUND OF THE INVENTION

[0003] Chronic obstructive pulmonary disease (COPD), consisting ofemphysema and chronic bronchitis, is the fourth leading cause of deathin the United States(1). Approximately 15 million Americans are affectedby COPD and there is an increasing incidence in women(2). Smoking is themajor risk factor for COPD and accounts for over 90% of cases seenworldwide. Despite the importance of the disease, there are no specifictherapies available to limit or prevent the slow, progressive,destructive changes observed in COPD(3).

[0004] Currently the major hypothesis for the pathogenesis of emphysemais the protease-antiprotease theory(4,5). This model suggests that animbalance between the levels of extracellular matrix degrading enzymesand their respective inhibitors damage the connective tissue matrixcomponents. of the lung. Studies over the past 30 years havedemonstrated differences in the protease levels in the lung of patientswith emphysema when compared to normal lung tissue(6). However, themolecular consequences of this finding have not been determined.

[0005] Although studies have demonstrated loss of the extracellularmatrix in the lung of patients with emphysema, an investigation as towhether cell death contributes to the pathogenesis of this disease hasnot been performed.

SUMMARY OF THE INVENTION

[0006] The present invention provides a method of treating or preventinga chronic obstructive pulmonary disease in a subject, comprisingadministering to said subject an amount of an agent effective to inhibitapoptosis of the subject's lung cells and thus treat or prevent chronicobstructive pulmonary disease in the subject. The present inventionprovides for a method of identifying a compound effective to treat orprevent a chronic obstructive pulmonary disease, comprising (a)contacting lung cells from a subject having a chronic obstructivepulmonary disease with the compound and measuring the level of apoptosisof the lung cells in the presence of said compound, (b) measuring thelevel of apoptosis of the lung cells from the same subject in theabsence of said compound, (c) comparing the level of apoptosis in step(a) with the level of apoptosis in step (b), wherein a higher level ofapoptosis in step (b) indicate that the compound is effective to treator prevent chronic obstructive pulmonary disease.

BRIEF DESCRIPTION OF THE FIGURES

[0007]FIG. 1. Light micrographs and TUNNEL staining of normal andemphysema lungs.

[0008] Panels A and B show hematoxylin and eosin staining of a normallung (A) and the lung from an emphysema patient (B). The emphysema lungexhibits thinning of the alveolar wall, and a pronouncedhypocellularity. The arrowhead identifies the nuclear pyknosis andfragmentation. In panel C-E, the TUNNEL reaction withfluorescein-incorporated dUTP was specifically observed in the emphysemalung specimen (D), but not in the normal counterpart (C). Panel Erepresents the reaction without TdT in the emphysema lung. The TUNNELreaction with biotinylated dUTP localizes apoptotic cells to both thealveolar surface and mesenchyme of the emphysema lung (G). Note somemacrophage-like cells contain TUNEL-reactive material in their cytoplasm(inset) The normal lung was not stained (F). Bar: 50 μm (A, B, F and G),100 μm (C-E) and 10 μm (inset in G).

[0009]FIG. 2. Nuclear disruption in the emphysema lung samples.

[0010] A. Ultrastructure of the alveolar septum of the emphysema lungtissue. Apoptotic cells (arrowhead) adjacent to a normal cell (★)illustrate cytoplasmic condensation and shrinkage, with condensation ofthe nuclear chromatin. Loss of cell-extracellular matrix contact is alsoobserved. Bar=2 μm. B. Isolated DNA from normal or emphysema lungtissues are electrophoresed on agarose gel (30 μg of DNA/lane) asdescribed in the methods section. In contrast to high Mr intact DNAisolated from the normal lung samples (lanes 1-3), DNA from theemphysema lung samples shows a characteristic DNA laddering on the gel(lanes 4-7). Lane M indicates size of DNA by 1 kb ladder DNA marker.

[0011]FIG. 3. Morphometry and apoptotic index in lungs.

[0012] A. Surface area and apoptotic index in groups of normal or mild,and moderate to severe emphysema patients. A significant difference isseen between normal-mild and moderate-severe grades for surface area andapoptotic index (p<0.01). B. An inverse correlation between surface areaand apoptotic index is observed by simple linear regression (r2=0.605).

[0013]FIG. 4. Caspase 3 and PARP cleavage in human lung tissue samples.

[0014] Tissue homogenates from normal (lanes 1-4) and emphysema lungs(lanes 5-8) were applied for the Western blot (120 g of totalprotein/lane) as described in METHODS. Lane 9 shows Jurkat cell lysatesstimulated by anti-Fas antibody as a positive control. Expression of thepro-form of caspase 3 (32 kba) is recognized by a monoclonal antibody tocaspase 3 in panel A. The active species of 17 and 12 kDa with a 24 kDaintermediate form are specifically detected by a rabbit polyclonalantibody in the emphysema samples (panel B). The degradation product ofPARP at 85 kDa is observed in the emphysema lung samples but not in thenormal lung samples (panel C).

[0015]FIG. 5. Detection of Bax and Bad and silver staining in lungsamples.

[0016] Normal (A, C and E) and emphysema lung specimens (B, D and F) aresubjected to immunostaining for Bcl-2 (A and B), Bax (C and D) and Bad(E and F). In the emphysema samples Bax is immunolocalized to thealveolar surface epithelial cells (arrowheads) (D), while bothmesenchymal (arrow) and alveolar surface epithelial cells (arrowhead)are recognized by the antibody to Bad (F). A high power view in panel Dinset demonstrates inclusion of anti-Bax antibody-reacted material in amacrophage-like cell. Bar: 50 μm (A, B, E and F), 25 μm (C and D) and 10μm (inset in D).

[0017]FIG. 6. Identification of sFRP1 expression in human lungs.

[0018] A, Differential Display was carried out with an upstreamarbitrary primer and a downstream anchor primer using total RNA samplesisolated from emphysema (Emp 1 and 2) and normal lungs (Nor 1 and 2).The arrowhead indicates the band for clone 1-41 which is detectable inthe emphysema but not normal lung samples. B, Cloned PCR fragments werescreened by dot blot hybridization using P³²-labeled emphysema andnormal lung first strand cDNA. The upper membrane was screened with theemphysema specific probe and demonstrated three positive clonesincluding 1-41 (arrow). The lower membrane was hybridized with thenormal lung probe. C, Total RNA isolated from emphysema (Emp 1-4) andnormal lungs (Nor 5-9) was subjected to RT-PCR using specific primersfor sFRP1, 2 and 3, respectively. No-RT is without reverse transcriptasein sample Emp 2. Exclusive amplification of sFRP1 in the emphysemasamples was observed. As an internal control for the presence of mRNA,RT-PCR was performed using a primer set for human GAPDH.

[0019]FIG. 7. sFrp1 expression in mouse lungs.

[0020] RT-PCR was undertaken for mouse samples. Mouse lung total RNA wasisolated from. cigarette-exposed (Em-sm) and collagenase transgenic mice(Em-MMPl), 14 dpc and 18 dpc embryo, newborn (NB) and normal adult mice(Nor). A reaction without reverse transcriptase in samples Em-MMPl isrepresented in the No-RT lane. Note sFrp1 expression in the emphysemamouse models (Em-sm and Em-MMPl) and embryos (14 and 18 dpc). As aninternal control for the presence of mRNA, RT-PCR was performed using aprimer set for mouse Gapdh.

[0021]FIG. 8. RT-PCR amplification of WNT and FZ mRNA in human lung.

[0022] Emphysema (Emp 1-4) and normal lung RNA (Nor5-9) were reversetranscribed by Superscript II using random oligomer. Primer pairsspecific for WNT5A, HZD2, HFZ6 and GAPDH were used for the PCR reaction.

[0023]FIG. 9. SFRP-1 leads to apoptosis of lung epithelial, endothelial& fibroblast cells in vitro.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention provides a method of treating or preventinga chronic obstructive pulmonary disease in a subject, comprisingadministering to said subject an amount of an agent effective to inhibitapoptosis of the subject's lung cells and thus treat or prevent chronicobstructive pulmonary disease in the subject.

[0025] In one embodiment, the agent inhibits apoptosis by inhibiting anapoptotic pathway. In another embodiment of the invention, the agentinhibits the apoptotic pathway by inhibiting expression of the sFRPgene.

[0026] In another embodiment of the invention, the sFRP gene comprises anucleic acid molecule comprising nucleotides having the sequence setforth in SEQ ID NO:1.

[0027] In another embodiment of the invention, the chronic obstructivepulmonary disease is emphysema.

[0028] In another embodiment of the invention, the chronic obstructivepulmonary disease is chronic bronchitis.

[0029] In another embodiment of the invention, the agent is selectedfrom a group consisting of an antisense molecule, a b chemokine, and aplant-derived composition.

[0030] In another embodiment of the invention, the antisense moleculecomprises nucleic acid having 8-30 nucleotides.

[0031] In another embodiment of the invention, the b chemokine is bchemokine I-309.

[0032] In another embodiment of the invention, the b chemokine is bchemokine TCA-3.

[0033] In another embodiment of the invention, the agent is the Herpessimplex virus ICP4.

[0034] The present invention provides for a method of identifying acompound effective to treat or prevent a chronic obstructive pulmonarydisease, comprising (a) contacting lung cells from a subject having achronic obstructive pulmonary disease with the compound and measuringthe level of apoptosis of the lung cells in the presence of saidcompound, (b) measuring the level of apoptosis of the lung cells fromthe same subject in the absence of said compound, (c) comparing thelevel of apoptosis in step (a) with the level of apoptosis in step (b),wherein a higher level of apoptosis in step (b) indicate that thecompound is effective to treat or prevent chronic obstructive pulmonarydisease.

[0035] In another embodiment of the invention, the level of apoptosis isdetermined by measuring DNA fragmentation or cleavage.

[0036] In another embodiment of the invention, the level of apoptosis isdetermined by measuring the expression of activated caspase 3.

[0037] In another embodiment of the invention, the level of apoptosis isdetermined by measuring the presence of poly (ADP ribose) polymerase.

[0038] In another embodiment of the invention, the level of apoptosis isdetermined by morphometric analysis.

[0039] In another embodiment of the invention, the level of apoptosis isdetermined by measuring Bcl-2 and Bad expression.

[0040] The invention also provides a method of treating or preventing achronic obstructive pulmonary disease in a subject, comprisingadministering to the subject an amount of an agent effective to inhibitexpression of a secreted Frizzled-related protein (sFRP) gene of thesubject's lung cells and thus treat or prevent chronic obstructivepulmonary disease in the subject. The chronic obstructive pulmonarydisease may be emphysema or chronic bronchitis.

[0041] The invention also provides an antibody capable of specificallybinding to sFRP, more specifically to sFRP-1. The antibody may be amonoclonal antibody or a polyclonal antibody. The antibody may behumanized.

[0042] The antibody may also be detectable. The antibody may be madedetectable by being labeled with a detectable marker. The the detectablemarker may be is a radioactive label or a calorimetric, or aluminescent, or a fluorescent marker.

[0043] The invention also provides a composition comprising the antibodyand an agent conjugated to the antibody. The agent may be a radioactiveisotope or toxin.

[0044] The invention also provides a method of determining whether asubject is afflicted with a chronic obstructive pulmonary disease whichcomprises: (a) obtaining a suitable sample from the subject; (b)contacting the suitable sample with the detectable antibody of claim 16so as to form a complex between the antibody and sFRP or fragmentthereof present in the sample; (c) removing any unbound antibody; and(d) detecting any antibody which is bound to any sFRP in the sample,wherein the presence of antibody indicates that the subject is afflictedwith the chronic obstructive pulmonary disease. The disiease may beemphysema. The suitable sample may be lung tissue.

[0045] In the method, the antigen bound by the antibody is detected byan immunoassay. The immunoassay may be ELISA, IFA, or Western blotting.

[0046] The invention also provides a kit for diagnosing chronicobstructive pulmonary disease comprising the labeled antibody. The kitmay furthe comprise a means for determining the level of sFRP orfragment thereof bound by an antibody. In the kit, the antibody may bebound to a support.

[0047] The invention also provides a method of inhibiting sFRP mediatedapoptosis of a cell which comprises introducing into the cell aneffective amount of the replicable vector which expresses an antisensemolecule to the gene encoding sFRP so as to thereby inhibit sFRPmediated apoptosis of the cell. The sFRP may be sFRP-1.

[0048] The invention also provides a method for evaluating in anon-human transgenic animal the potential therapeutic effect of an agentfor treating chronic obstructive pulmonary disease in a human, whichcomprises: (a) providing an agent to a transgenic non-human animalhaving chronic obstructive pulmonary disease;(b) determining thetherapeutic effect of the agent on the transgenic non-human animal bymonitoring sFRP expression, wherein a decrease in sFRP indicates thatthe agent would have a potential therapeutic effect on chronicobstructive pulmonary disease in a human. The animal may be a mammal.The non-human animal may be a mouse, a rat, a sheep, a dog, a primate,or a reptile.

[0049] The invention also provides method of detecting a chronic obstr

[0050] a) obtaining a suitable sample of mRNA from the subject; b)contacting the mRNA sample under hybridizing conditions with a labelednucleic acid probe which: (1) is at least 15 nucleotides in length and(2) hybridizes specifically to a nucleic acid having a sequence which iscomplementary to a sequence present in the sequence set forth in SEQ IDNO. 2; c) removing any unbound labeled nucleic acid probe; and d)detecting the presence of labeled nucleic acid probe hybridized to themRNA so as to thereby detect chronic obstructive pulmonary disease inthe subject.

[0051] In the method, the mRNA may be from lung tissue of the subject.

[0052] The invention also provides method of detecting chronicobstructive pulmonary disease in a subject which comprises: a) obtaininga suitable sample of mRNA from the subject; b) reverse transcribing themRNA to generate a single-stranded cDNA; c) contacting thesingle-stranded cDNA under hybridizing conditions with a labeled nucleicacid probe which: 1) is at least 15 nucleotides in length; and 2)hybridizes specifically to a nucleic acid having a sequence set forth inSEQ ID NO:2; d) removing any unbound labeled nucleic acid probe; and e)detecting the presence of labeled nucleic acid probe hybridized to thecDNA so as to thereby detect detect chronic obstructive pulmonarydisease in the subject.

[0053] The invention also provides method of detecting chronicobstructive pulmonary disease in a subject which comprises: a) obtaininga suitable sample of mRNA from the subject; b) generating adouble-stranded mRNA-cDNA duplex from the mRNA; c) contacting the duplexfrom (b) with one primer having a sequence which is complementary to aportion of the sequence set forth in SEQ ID NO:2 and a second primerhaving a sequence which comprises a different portion of the sequenceset forth in SEQ ID NO:2; d) amplifying the nucleic acid from (c) usinga polymerase chain reaction to obtain an amplification product; e)contacting the amplification product of (d) under hybridizing conditionswith a labeled nucleic acid probe which: 1) is at least 15 nucleotidesin length; 2) hybridizes specifically to a nucleic acid having asequence set forth in SEQ ID NO. 2; f) removing any unbound labelednucleic acid probe; and g) detecting the presence of labeled nucleicacid probe hybridized to the amplification product so as to therebydetect chronic obstructive pulmonary disease in the subject.

[0054] As used herein, the term “apoptosis” means programmed cell deathor cell death caused by an active process of gene-directed cellularself-destruction and characterized by the rapid condensation of the cellwith preservation of membranes, the compaction of chromatin, and DNAcleavage and fragmentation. The mechanism of apoptosis is described indetail in Granville D. J., et al. (1998) “Apoptosis: Molecular aspectsof cell death and disease” Lab. Invest., 78:893-913 and the content ofGranville D. J., et al. is fully incorporated in its entirety byreference.

[0055] As used herein, “Wnt gene” represents genes encoding Wntglycoproteins which serve as inducers of cellular proliferation,migration, differentiation and tissue morphogenisis during normaldevelopment.

[0056] The term “FRP” means Frizzled-related Proteins which contain aregion homologous to a putative Wnt-binding domain of Frizzleds andwhich serve as antagonists of Wnt action. The term “sFRP” means secretedFrizzled-related Proteins. EP 0 879 885 discusses a human gene similarto a secreted murine protein sFRP-1. One of the Frizzled-relatedProteins or the secreted Frizzled-related Proteins is a polypeptidehaving an am (SEQ ID NO:1)MGIGRSEGGRRGALGVLLALGAALLAVGSASEYDYVSFQSDIGPYQSGRFYTKPPQCVDIPADLRLCHNVGYKKMVLPNLLEHETMAEVKQQASSWVPLLNKNCHAGTQVFLCSLFAPVCLDRPIYPCRWLCEAVRDSCEPVMQFFGFYWPEMLKCDKFPEGDVCIAMTPPNATEASKPQGTTVCPPCDNELKSEAIIEHLCASEFALRMKIKEVKKENGDKKIVPKKKKPLKLGPIKKKDLKKLVLYLKNGADCPCHQLDNLSHHFLIMGRKVKSQYLLTAIHKWDKKNKEFKNFMKKM KNHECPTFQSVFK

[0057] The term “sFRP genes” means DNA molecules encodingFrizzled-related Proteins. One of the sFRP genes is a nucleic acidcomprising nucleotides having the sequence as set forth in SEQ ID NO:2as follows: CCTGCAGCCT CCGGAGTCAG TGCCGCGCGC CCGCCGCCCC GCGCCTTCCT (SEQID NO:2) GCTCGCCGCA CCTCCGGGAG CCGGGGCGCA CCCAGCCCGC AGCGCCGCCTCCCCGCCCGC GCCGCCTCCG ACCGCAGGCC GAGGGCCGCC ACTGGCCGGG GGGACCGGGCAGCAGCTTGC GGCCGCGGAG CCGGGCAACG CTGGGGACTG CGCCTTTTGT CCCCGGAGGTCCCTGGAAGT TTGCGGCAGG ACGCGCGCGG GGAGGCGGCG GAGGCAGCCC CGACGTCGCGGAGAACAGGG CGCAGAGCCG GCATGGGCAT CGGGCGCAGC GAGGGGGGCC GCCGCGGGGCCCTGGGCGTG CTGCTGGCGC TGGGCGCGGC GCTTCTGGCC GTGGGCTCGG CCAGCGAGTACGACTACGTG AGCTTCCAGT CGGACATCGG CCCGTACCAG AGCGGGCGCT TCTACACCAAGCCACCTCAG TGCGTGGACA TCCCCGCGGA CCTGCGGCTG TGCCACAACG TGGGCTACAAGAAGATGGTG CTGCCCAACC TGCTGGAGCA CGAGACCATG GCGGAGGTGA AGCAGCAGGCCAGCAGCTGG GTGCCCCTGC TCAACAAGAA CTGCCACGCC GGGACCCAGG TCTTCCTCTGCTCGCTCTTC GCGCCCGTCT GCCTGGACCG GCCCATCTAC CCGTGTCGCT GGCTCTGCGAGGCCGTGCGC GACTCGTGCG AGCCGGTCAT GCAGTTCTTC GGCTTCTACT GGCCCGAGATGCTTAAGTGT GACAAGTTCC CGGAGGGGGA CGTCTGCATC GCCATGACGC CGCCCAATGCCACCGAAGCC TCCAAGCCCC AAGGCACAAC GGTGTGTCCT CCCTGTGACA ACGAGTTGAAATCTGAGGCC ATCATTGAAC ATCTCTGTGC CAGCGAGTTT GCACTGAGGA TGAAAATAAAAGAAGTGAAA AAAGAAAATG GCGACAAGAA GATTGTCCCC AAGAAGAAGA AGCCCCTGAAGTTGGGGCCC ATCAAGAAGA AGGACCTGAA GAAGCTTGTG CTGTACCTGA AGAATGGGGCTGACTGTCCC TGCCACCAGC TGGACAACCT CAGCCACCAC TTCCTCATCA TGGGCCGCAAGGTGAAGAGC CAGTACTTGC TGACGGCCAT CCACAAGTGG GACAAGAAAA ACAAGGAGTTCAAAAACTTC ATGAAGAAAA TGAAAAACCA TGAGTGCCCC ACCTTTCAGT CCGTGTTTAAGTGATTCTCC CGGGGGCAGG GTGGGGAGGG AGCCTCGGGT GGGGTGGGAG CGGGGGGGACAGTGCCCGGG AACCCGTGGT CACACACACG CACTGCCCTG TCAGTAGTGG ACATTGTAATCCAGTCGGCT TGTTCTTGCA GCATTCCCGC TCCCTTTCCC TCCATAGCCA CGCTCCAAACCCCAGGGTAG CCATGGCCGG GTAAAGCAAG GGCCATTTAG ATTAGGAAGG TTTTTAAGATCCGCAATGTG GAGCAGCAGC CACTGCACAG GAGGAGGTGA CAAACCATTT CCAACAGCAACACAGCCACT AAAACACAAA AAGGGGGATT GGGCGGAAAG TGAGAGCCAG CAGCAAAAACTACATTTTGC AACTTGTTGG TGTGGATCTA TTGGCTGATC TATGCCTTTC AACTAGAAAATTCTAATGAT TGGCAAGTCA CGTTGTTTTC AGGTCCAGAG TAGTTTCTTT CTGTCTGCTTTAAATGGAAA CAGACTCATA CCACACTTAC AATTAAGGTC AAGCCCAGAA AGTGATAAGTGCAGGGAGGA AAAGTGCAAG TCCATTATCT AATAGTGACA GCAAAGGGAC CAGGGGAGAGGCATTGCCTT CTCTGCCCAC AGTCTTTCCG TGTGATTGTC TTTGAATCTG AATCAGCCAGTCTCAGATGC CCCAAAGTTT CGGTTCCTAT GAGCCCGGGG CATGATCTGA TCCCCAAGACATGTGGAGGG GCAGCCTGTG CCTGCCTTTG TGTCAGAAAA AGGAAACCAC AGTGAGCCTGAGAGAGACGG CGATTTTCGG GCTGAGAAGG CAGTAGTTTT CAAAACACAT AGTTA

[0058] As used herein, the phrase “Chronic Obstructive PulmonaryDisease” means a process characterized by the presence of chronicbronchitis or emphysema that may lead to the development of airwaysobstruction, both reversible airways obstruction and irreversibleairways obstruction. “Chronic obstructive pulmonary disease” includeschronic bronchitis, emphysema, and asthma.

[0059] As used herein, “inhibitors of cell apoptosis” includes, but notlimited to, antisense compounds, such as described in Bennett, et al.,U.S. Pat. No. 5,958,772, plant-derived compositions as described inBathurst, et al., U.S. Pat. Nos. 5,620,885, 5,567,425, 5,624,672,5,759,548 and 5,635,187, b chemokines, such as b chemokine I-309 and bchemokine TCA-3, as describe in Damme, et al., U.S. Pat. No. 5,824,551.“Inhibitors of cell apoptosis” also includes, but not limited to, andHerpes simplex virus ICP4 as described in Leopardi, et al., U.S. Pat.No. 5,876,923.

Experimental Details EXAMPLE 1 Apoptosis in Human Emphysema Lungs,Implications for Novel Therapeutic Strategies

[0060] Lung Samples:

[0061] Human lung tissue was obtained between 1995-1999 from a total of19 patients at Columbia Presbyterian Medical Center (IRB #X042 1) asfollows: 14 samples were obtained from patients with emphysema whounderwent lung transplant and five samples from patients who underwentlung volume reduction procedures. Samples from six normal lungs wereused as controls. The six normal lung samples were obtained from donorlungs harvested from transplant but not used due to recipientcomplications or from accidental death victims. All of the emphysemasamples were taken from patients who reportedly had stopped smoking forat least three months prior to harvesting the tissues. Cellular deathwas evaluated morphologically, histochemically and biochemically.Western blot analysis was performed to identify the presence of activecaspase 3 and poly(ADP-ribose) polymerase in the emphysema tissue.Expression of the anti-apoptotic Bcl-2 protein, and its pro-apoptoticcounterparts Bax and Bad were also determined throughimmunohistochemistry.

[0062] Histological Examination:

[0063] After surgical excision, lungs were immediately fixed in 10%neutral buffered formalin for about 16 hours at 4° C. and embedded inparaffin-wax. Every sample was examined histologically in a blindedfashion for the presence of emphysema, fibrosis and inflammation andsamples with pathological evidence of inflammation indicative of ongoinginfection or neoplastic changes were excluded from this study. Sections(3 μm) were stained by silver impregnation for collagen fibrils(7,8).Immunohistochemical staining was performed using mouse IgG specific tohuman Bad (clone B31420, 10 g/ml) or Bcl-2 (clone B31420, 10 g/ml)(Transduction Laboratories, Lexington, Ky.) and rabbit polyclonalantibody to human Bax (clone 13666E, dilution×1,000. PharMingen, SanDiego, Calif.). After incubation with biotinylated horse IgG to mouseIgG or goat IgG to rabbit IgG (Vector Laboratories, Burlingame, Calif.)and an avidin-biotin-peroxidase complex (DAKO, Glostrup, Denmark), colorwas developed with 3,3′-diaminobenzidine tetrahydrochloride. Fortransmission electron microscopy, tissues were cut into small pieces andfixed in 2.5% glutaraldehyde followed by 2% osmium tetroxide at 4° C.and processed to ultrathin sections for the electron microscope (1200 EXII, 80 KV, Jeol, Sundbyberg, Sweden).

[0064] In Situ Labeling of DNA Cleavage:

[0065] Formalin-fixed specimens were subjected to oligonucleosomalfragment labeling of DNA by terminal deoxynucleotidyl transferase(TdT)-mediated X-dUTP nick end labeling (TUNNEL), using DeadEndColorimetric Apoptosis Detection System (Promega, Madison, Wis.) forstreptavidin horseradish peroxidase-diaminobenzidine detection and InSitu Cell Death Detection Kit (Boehringer Mannheim, Indianapolis, Ind.)for fluorometric detection of apoptotic cells. These reactions wereundertaken according to the manufacture's instructions. The percentageof TUNNEL reactive cells to total cells (apoptotic index) was measuredin three different, areas in each specimen at 40-fold magnificationusing light microscopy. The significance of difference in the apoptoticindex between normal-mild emphysema and moderate-severe emphysema wasdetermined by a Mann-Whitney U test. As a positive control, lungspecimens were treated with RNase-free DNase I (Boehringer Mannheim)followed by TdT reaction. As a negative control, TdT was omitted fromthe reactions.

[0066] Morphometric Analysis:

[0067] Tissue sections were stained with hematoxylin and eosin and themean linear intercept and internal surface area were calculatedaccording to established methods (9-11) using a light microscope linkedto a Macintosh computer and Adobe Photoshop imaging software. Arectangular grid of dots at approximately 1 mm intervals was applied to10 different areas in each section. From a random starting position onthe grid, sequential and spaced images were digitally recorded foranalysis. A test system was randomly superimposed upon each image.Horizontal lines were used to count alveolar surface intersections.Endpoints were used to calculate alveolar volumes. Results were analyzedwith one-way analysis of the variance and simple linea between surfacearea and apoptotic index.

[0068] DNA Fragmentation:

[0069] Four emphysema and three normal lung tissues samples (100 mg oftissue wet weight) were digested with 0.1 mg/ml of Proteinase K for ˜16h in 1.2 ml of digestion buffer (10 mM Tris-HCl, 0.1 M NaCl, 25 mM EDTA,0.5% SDS, pH 8.0). After protein extraction with phenol-chloroformisoamylalcohol and dialysis against 10 mM Tris-HCl, 1 mM EDTA, pH 8.0,samples were incubated with 1 μg/ml of RNase A for 1 h at 37° C. anddialyzed in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0, at 4° C. For detection ofDNA fragmentation, 30 μg of isolated DNA were size fractionated on 1.4%agarose gel containing 0.1 μg/ml of ethidium bromide.

[0070] Protein Preparation and Analysis:

[0071] Tissue homogenates of six emphysema and five normal lungs wereprepared in 20 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.02% NaN3, 1% NP-40, 1mM PMSF, 2 mM N-ethylmaleimide, 10 μg/ml of leupeptin, 1 μg/ml ofaprotinin, 10 μg/ml of pepstatin A, 10 μM E-64 and 1 mM EDTA. Proteins(120 μg) in the homogenate was size fractionated on SDS-PAGE underreducing conditions and electrotransferred onto a nitrocellulosemembrane (Trans-Blot, BioRad, Hercules, Calif.). For immunologicaldetection of proteins, Western blot was performed using mouse IgG tohuman caspase 3, clone C31720, 0.4 μg/ml (Transduction Laboratories) orpolyclonal rabbit anti-human caspase 3 (PharMingen, Clone 67341A, 1μg/ml) as previously described (12). Rabbit antibody against theproteolytic fragment of poly(ADP-ribose) polymerase (PARP) (Promega,Clone G734, 0.35 μg/ml) was also used.

[0072] Fluorometric Assay:

[0073] Four emphysema and four normal lung tissues were homogenized in20 mM Tris-HCl, pH 7.4, 10 mM Na2P2O7, 100 mM NaF, 2 mM NaVO4, 5 mMEDTA, 1 mM PMSF, 10 μg/ml aprotinin, and 1% NP-40. After removal ofinsoluble materials, caspase 3 activity was quantified by thefluorometric assay using specific synthetic peptide substrate(Ac-DEVD-AMC, PharMingen) as previously described(13).

[0074] Morphological and Biochemical Detection of Apoptosis:

[0075] In normal lungs, the alveolar wall consists of three tissuecomponents including the surface epithelium, supporting connectivetissue and blood vessels (FIG. 1A). The supporting tissue forms a layerbeneath the epithelium and surrounding the blood vessels of the alveolarwall. In contrast, extensive loss of the alveolar architecture in theemphysema lungs is associated with hypocellularity and thinning of theremaining alveolar wall (FIG. 1B). Within the emphysema lung samples,cells were morphologically characteristic of cells undergoing apoptosisexhibiting convolution of nuclear outlines (FIG. 1B, arrow). Thesenuclear changes were observed in cells throughout the sample and not infocal regions as is seen in necrosis(14). The apoptotic cells includedendothelial cells, epithelial cells and fibroblasts. Neutrophilinfiltration into the alveolar space or the alveolar septa wasnegligible.

[0076] In Situ Detection of DNA Cleavage:

[0077] To confirm the presence of apoptosis in the emphysema lungsamples, two different TUNEL reactions were carried out. The firstreaction using fluorescein-conjugated nucleotide exhibited little or nolabeling in the normal lung tissue samples (FIG. 1C) while many cellswith intense labeling were present throughout the emphysema tissue (FIG.1D). Another TUNEL assay was performed using biotinylated nucleotide toidentify the apoptotic cell type under the light microscope. Normal lungspecimens did not react to TUNEL staining (FIG. 1F). In contrast tonormal tissues, emphysema sections were TUNEL positive, however, therewas no prevalent cell type. Throughout the emphysema lung specimen,alveolar and mesenchymal cells both exhibited positive TUNEL staining(FIG. 1G). In the emphysema tissue section, 6.1±3.5% (mean±1 S.D.) ofcells were labeled (varied in cases from 1.3±0.3 to 12.2±3.5), whereasvery few cells were positive in the normal lung samples (0.1±0.1%)(p<0.01). Several macrophage-like cells were TUNEL-reactive in theircytoplasm characteristic of phagocytosis of apoptotic cell bodies (FIG.1G, inset).

[0078] Ultrastructural analysis of the emphysema lung tissuedemonstrated morphological changes consistent with apoptosis in severalcell types. The most prominent feature seen was cytoplasmic condensationand vacuolization, chromatin condensation and connective tissuedegradation. A representative example can be seen in FIG. 2A with twoapoptotic cells (arrows) in close apposition to a healthy cell; note thecytoplasmic condensation and nuclear condensation in the apoptotic cellwith irregularities in the cell shape.

[0079] The presence of apoptosis in the emphysema lung samples wasconfirmed using biochemical analysis of DNA laddering (FIG. 2B).Electrophoresis of DNA isolated from emphysema tissues demonstrateddegradation into small laddering fragments of multiples of approximately180 bp subunit in contrast to the intact high Mr size seen in the normalsamples.

[0080] Correlation of Morphometric Measurements with Apoptotic Index

[0081] Morphometric studies were performed on lung tissue examined inthe above studies and surface area was calculated for each sample.Samples were divided into groups according to the severity of emphysemabased on surface area measurements. There was a statisticallysignificant association between the apoptotic index and emphysemaseverity (p<0.01) (FIG. 3A). In addition, through regression analysisthe apoptotic index was shown to inversely correlate with the surfacearea demonstrating an increase in apoptosis with decreased surface area(FIG. 3B)

[0082] Caspase Expression, and Activity in Lung Homogenates:

[0083] Aspartate-directed cysteine proteases, caspases, play a pivotalrole in execution of the apoptotic pathway, but not in necrosis(15). Inorder to detect caspase expression, we subjected tissue homogenatesdirectly to Western blot analysis. As shown in FIG. 4A, pro-caspase 3(32 kDa) was detected in both normal and emphysema lung homogenates withno clear difference in expression levels in these samples. This resultwas confirmed by reactivity with a rabbit polyclonal antibody againstcaspase 3 (data not shown). The activated subunits of caspase 3 (p17 andp12) were, however, only detected in the emphysema lung homogenates(FIG. 4B). In addition, an antibody that specifically reacts to theproteolytic fragment of PARP, a substrate of caspase 3, demonstratedreactivity in the emphysema lung tissue homogenates but not in normallung tissues (FIG. 4C). Using a fluorogenic synthetic peptide substratecaspase 3 activity was detected in the emphysema lung homogenates andnot in normal lung homogenates (data not shown).

[0084] Bcl-2 and Bad Expression and Degradation of Collagen Fibrils:

[0085] Recent studies indicate that the ratio of Bax protein expressionto Bcl-2 expression is increased in apoptotic cells, especially whencells loose contact with extracellular matrix attachment(16, 17).Immunohistochemical analysis to detect the expression of Bcl-2, Bax andBad was performed. Although Bcl-2 was not detected in either normal oremphysema lung tissue (FIGS. 5A and 5B), Bax and Bad reactivity was seenonly in the emphysema lung samples (FIGS. 5C-F). Bax staining waslocalized to the epithelial cells in the emphysema lung samples (FIG.5D, arrow, inset), whereas Bad staining was localized randomly to theepithelial and mesenchymal cells (FIG. 5F). This immunolocalization ofBad is consistent with the pattern of the TUNEL reaction. The normallung tissue was negative for Bad immunostaining (FIG. 5E).

[0086] Discussion

[0087] In the present study, we examined morphological changes, DNAfragmentation, caspase activation and connective tissue degradation inhuman emphysema and normal lung tissues. Chronic obstructive pulmonarydisease (COPD) is believed to be caused by exposure to cigarette smoke.However, the cellular mechanisms responsible for the progressivedeterioration of respiratory function in COPD remain unclear and appearto result from architectural destruction including cellular disruptionthat may be associated with apoptosis. Our results demonstrate extensivecell death through apoptosis in the emphysema lungs.

[0088] Emphysema is postulated to develop from disruption of theextracellular matrix through an imbalance between proteases andantiproteases(6, 18). In the present study, we demonstrated for thefirst time that there is extensive cell death by apoptosis incombination with connective tissue degradation in the human emphysemalung. In this chronic disea which could be accounted for by apoptosis.

[0089] Two different mechanisms, i.e., necrosis and apoptosis areobserved in cellular death. The two processes can be distinguished bydistinct morphological features. Necrotic cells exhibit severalcharacteristic features such as cellular swelling and rupture of theplasma membrane, while the nucleus remains relatively intact. Necrosisis usually associated with an inflammatory reaction which develops inthe adjacent viable tissue in response to the release of cellulardebris. On the other hand, cell shrinkage and blebb and intactcytoplasmic organelles morphologically characterize apoptotic cells(14). The morphological features of the emphysema lung cells in thisstudy are consistent with apoptosis. In addition, we found evidence ofDNA fragmentation in lung samples from patients with emphysema on thebasis of both in situ end labeling and gel electrophoresis. Thehistological analysis and the TUNEL assay demonstrated no specificity incell-types undergoing apoptosis. However, these observations are basedon tissue samples at the end stage of the disease. We frequentlyobserved TUNEL-positive material-containing macrophages in the emphysemaspecimens, suggesting a role for alveolar macrophages as a scavenger ofapoptotic cells.

[0090] Caspase-3 processing into active species in the emphysema lungtissue, but not in the normal lung, strongly supports our observation ofongoing apoptosis in the emphysema lungs. Exclusive caspase 3 activityagainst a synthetic peptide in the emphysema samples confirms thisobservation. The sequence of caspase activation is an indispensableprocess in the apoptosis pathway(19). Caspase 3 functions down-stream ofcell damage in the apoptotic pathway and has a pivotal role in targetingmolecules for proteolysis. Proteolysis of PARP by caspase 3 is aspecific event that occurs during apoptosis(19). Detectable degradationof PARP into an 85 kDa fragment was observed in the emphysema tissuesamples indicating caspase activity in the emphysema tissues but not inthe normal lungs.

[0091] The close correlation of apoptosis with the morphologicalparameters of the disease was demonstrated in this study. Although theapoptotic index was variable between patient samples, statisticalanalysis demonstrates that the increase in apoptotic cell deathassociates with more severe structural destruction of the lung. Whencomparing the apoptotic index with morphometric measurements ofemphysema this study strongly demonstrates a correlation betweenapoptosis and severity of disease and emphasizes the potentialinvolvement of apoptosis in emphysema progression. The direct mechanismof apoptosis in human studies is not easily identifiable, however,intuitively the disruption of the extracellular matrix through the knownprotease-antiprotease imbalance could lead to induction of the cellulardeath program. The presence of apoptosis in the lung does not negate therole of proteases in the pathogenesis of the disease and may be acontinuum in the process of destruction. The failure of the lung tomaintain its cellular architecture in the presence of excess proteasesmay ultimately lead to the induction of apoptosis. It is known thatexpression of pro-apoptotic Bax family members is increased when cellsare dying through depletion of cell adhesion to the extracellularmatrix(16, 17) The Bax family members counteract Bcl-2 function andtrigger caspase activation. Our immunostaining data suggests that thereis an increase of Bax protein staining in contrast to Bcl-2 in theemphysema tissue. It is known that in emphysema tissue there isextensive loss of the extracellular matrix leading to massive connectivetissue damage in the emphysema alveolus(20). Furthermore, TUNNELpositive staining was seen in a transgenic mouse model of emphysema (7)as compared to the wild-type litter mates (Imai et al, unpublishedresults). This transgenic model develops emphysema as a result ofcollagenase disruption of the extracellular matrix.(7) Immunoreactivityto anti-Bad antibody was also increased in the emphysema lungs. Althougha role for increased Bad expression has not been defined, Bad is knownto counteract Bcl-2 induced apoptosis. Therefore, the combination ofincreased Bax and Bad staining, increased apoptosis in the transgenicmouse and loss of the extracellular matrix in emphysema leads us tohypothesize that connective tissue degradation in the alveolar septaabrogates the cell-matrix attachment and contributes to induction ofapoptosis.

[0092] Recently, we identified emphysema specific expression of secretedfrizzled-related protein using a differential display assay(21). Thismolecule inhibits Wnt binding to its cell surface receptor frizzled.Although targeting of the Wnt signal in mammals is not well defined,expression of the exogenous Wnt gene in cultured cells promotes cellularproliferation(22). Interestingly, secreted frizzled-related protein wasalso identified as an apoptosis-inducing protein in cultivated cells(23,24). Thus, the inhibition of the Wnt signaling pathway could possibly beinvolved in the apoptosis seen in the emphysema lung.

[0093] In the present study, we demonstrate for the first time thatextensive apoptosis is occurring in emphysema lung. This is anintriguing novel mechanism in which to explain the destruction of thelung during progression of the disease. This is the first demonstrationin emphysema that cellular loss in addition to matrix loss plays a rolein the disease process. Recent studies demonstrated that apoptosis isoccurring in a variety of chronic human diseases includingneurodegenerative disease, heart failure, atherosclerosis, and viraldiseases(19, 25). In several of these diseases, anti-apoptotic agentsare expected to treat patients or slow disease progression and many ofthose agents are under evaluation and could potentially be applied toemphysema.

[0094] Conclusions

[0095] Lungs from all emphysema samples, but not normal controls, showedevidence of DNA fragmentation as determined by TUNNEL assays. Inagreement with the positive TUNNEL assays, the emphysema lung samplesalso exhibited DNA indicative of cells undergoing apoptosis. Westernblot analysis exhibited expression of activated caspase 3 and thepresence of a specific cleavage product of poly(ADP-ribose) polymerase.Finally, immunohistochemistry demonstrated increased expression ofpro-apoptotic molecules Bax and Bad in the emphysema lung samples withno increase in BCL-2.

[0096] The novel demonstration of apoptosis in the emphysema lungsuggests that programmed cell death contributes to the progressive lossof respiratory function in this disease. Therefore, disrupting theapoptotic pathway could be an alternative approach to therapy in humans.

EXAMPLE 2 Activation of an Embryonically Expressed Gene in PulmonaryEmphysema. Identification of the Secreted Frizzled-Related Protein.

[0097] Differential display analysis was performed on lung tissue toidentify genes expressed in emphysema but not in normal lung. Secretedfrizzled-related protein 1 (sFRP1), an inhibitor of wnt signaling, wasfound to be expressed in emphysema but not in normal lung tissue. Othermembers of the sFRP family did not demonstrate differential expressionin lung tissue. Expression of the mouse homologue, sFrp1, was alsodetectable only in emphysema and not in normal lungs of mice. Finally,embryonic-specific expression of sFrp1 suggests that the Wnt signalingpathway is normally involved in lung development. The novelidentification of an embryonic gene activated in emphysema providesinsight into the pathophysiological changes in this disease.

[0098] In this Example, RNA fingerprinting (27) was used to compareexpressed genes between emphysema and normal human lung tissue (FIG.6A). After subcloning 30 gene candidates that were differentiallyexpressed, a dot blot hybridization was performed using the first strandcDNA from normal and human emphysema lung samples as probes forsecondary screening. Nine of these clones led to one-sided signalsbetween the two probes (FIG. 6B). Five clones were expressed only in theemphysema lung sample and four clones were expressed only in the normallung sample. These clones were subjected to DNA sequence analyses. Basedon sequence data, reverse transcription (RT)-PCR was performed to verifythe expression pattern of these genes. Through RT-PCR, clone 1-41 wasshown to be expressed in the emphysema lung samples but not detected inthe normal lung (FIG. 6C). None of the other clones demonstrated such anabsolute difference in their pattern of “expression (data not shown) andthus these clones were not analyzed further.

[0099] A search of the Genbank database revealed partial homology ofclone 1-41 to mouse and bovine secreted frizzled-related protein 1(sFrp1).(28, 29) In addition, a search of the expressed sequence tag(EST) database localized this sequence to the chromosomal region of8p11-12 (A002C42, stSG3941), between D8S1791-D8S268 (NCBI accession no.:W21306, H29416, H29323, H16861, H16753 and H12000). This chromosomalsite is identical to the chromosomal site of the human sFRP1 gene.(26)5′RACE was performed to identify the upstream sequence of clone 1-41 anda 1.1 kb cDNA fragment was obtained. The upstream 530 bp of the 5′-endsequence of clone 1-41 demonstrated 97% identity to the 3′-end of thehuman sFRP1 gene.(26) Therefore, we considered clone 1-41 to be the3′-end of the human sFRP1 gene and used the open reading frame sequencefrom human sFRP1 in subsequent experiments.

[0100] sFRP1 belongs to a gene family of five molecules,sFRP1˜sFRPS.(29-32, 26) Through RT-PCR analysis the expression patternof the sFRP family members were examined. Only sFRP1 was found to beexpressed in emphysema and not in the normal lung tissue. sFRP2 andsFRPS were not detected in any of the samples (FIG. 6C). sFRP3 wasdetected in only one of four emphysema lungs and in none of the normallung samples (FIG. 6C). sFRP4 was detected in all of the emphysema andnormal lung samples as demonstrated in previous studies (data notshown).(26, 30, 31, 33)

[0101] Animal models of emphysema have been developed which help inunderstanding the pathogenesis of emphysema.(34-35) Transgenic micewhich express human interstitial •collagenase in the lung developemphysema strikingly similar to the human disease.(7) The smoke-exposedmouse has also been used as a mouse model of emphysema.(35) Althoughexpression of the mouse sFrp1 homologue was not observed in the normaladult mouse lung, both transgenic and smoke-exposed mice expressed sFrp1(FIG. 7). The mouse homologue for sFrp3 was not amplified in any samplesexamined (data not shown).(28)

[0102] To rule out the possibility that sFrp1 was induced solely throughnonspecific injury to the lung, intraperitoneal injections oflipopolysaccharide (LPS) were given to mice. Systemic administration ofLPS results in increasing proinflammatory cytokines in the lung andinduction of acute phase inflammatory proteins such as haptoglobin.(36)LPS injection in the mice did not upregulate sFrp1 expression in thelung, but, as expected, haptoglobin expression was induced (data notshown). This result indicates that induction of sFrp1 is relatedspecifically to the pathological changes which occur in emphysema.

[0103] Expression of sFrp1 in the embryonic developmental lung wasexamined. Although re-expression of developmentally regulated genes hasbeen found in a variety of diseases(37), this has never been observed ornoted in emphysema. Specific amplification of the sFrp1 PCR product wasobserved in the embryonic mouse lungs (14 and 18 dpc) in contrast to nodetectable expression in the newborn and adult lungs (FIG. 7).

[0104] The biological activity of sFRPs is closely related to thefunction of the Wnt family of proteins. The basic structure of sFRP ishomologous to the extracellular Wnt-binding domain of Frizzled (FZ), theWnt cell surface receptor(32), but lacks the seven transmembranespanning sequence that anchors the protein to the cell surface andtransmits Wnt signaling-activities(32). Therefore, sFRPs are believed toinhibit the Wnt-inducible signaling pathway by antagonizing Wnt-FZbinding in the extracellular milieu(32). The interaction of eachspecific sFRP with its respective Wnt molecule has not been defined.Therefore we wished to examine if any Wnt molecules were. expressed inthe lung. The Wnt family is divided into two classes according to theirability to transform mammary epithelial cells and cause axis-inductionin Xenopus (Wntl class and WntSA class).(32) In this study, WNT1 and 8B(Wntl class) mRNA was not amplified in human lungs. However, the WNT5Atranscript was detected in all of the emphysema samples and two of thefive normal samples (FIG. 8). These studies on Wnt lead to theconclusion that the WntSA class is expressed in the lung and suggestthat this class of proteins may be a potential target of sFRP1inhibition in emphysema.

[0105] Four members of the human FZ family have been isolated, HZD2,HZD3, HFZ5 and HFZ6.(31) Expression of these genes in the lung has notyet been defined. Through RT-PCR analysis, HZD2 was found to beexpressed in three of the four emphysema and four of the five normallungs (FIG. 8). HFZ6 was present in all of the samples (FIG. 8). HZD3and HFZ5 were not amplified by the PCR reactions (data not shown). Thedemonstration of WNT and FZ (HZD2 and HFZ6) within the lung confirmsthat the signaling machinery for the sFRP is indeed present within thelung.

[0106] Expression of sFRP1 was observed exclusively during embryogenesisin the lung. The identification of sFRP1 in the embryonic lung will leadto future studies which identify the cell type of expression and theinteractions with specific FZ and Wnt family genes in lung development.Intriguingly, sFRP1 is expressed in the adult tissue when the lung isinjured in emphysema. The specific elevated expression of sFRP1 inemphysema was not a generalized phenomenon for all members of this genefamily, because the expression sFRP2-sFRP5 was not altered betweenemphysema and normal lung samples. The presence of sFRP1 in emphysemademonstrates for the first time developmental gene re-expression in thisdisease. The Wnt signaling pathway plays a pivotal role duringembryogenesis and in the state of tissue injury in emphysema thisprocess could be recapitulated. The re-expression of developmentallyregulated genes in this disease may reflect a protective role for sFRP1during the repair process. Emphysema is characterized by extensivechronic destruction of the lung architecture and is associated with avariety of tissue reactions. Investigators have focused primarily on therole of proteases and anti-proteases in this disease process.(4)However, this study demonstrates a novel molecular pathway involving Wntsignaling in the pathophysiology of emphysema. Further avenues ofresearch will define the link between sFRP1 expression and the specificWnt signaling pathway and elucidate the role of this pathway in lungdisease.

[0107] Methods.

[0108] Lung Samples.

[0109] Six cases of human emphysema lung tissue were obtained atColumbia Presbyterian Medical Center from recipient lungs duringtransplantation or lung volume reduction procedures. The majoretiological factor for emphysema in these patients was cigarettesmoking. All samples were taken from patients who reportedly stoppedsmoking for at least three months (mean age±S.D., 47±11 years). Fivenormal lungs were obtained from donor lungs harvested for transplant butnot used due to recipient complications. All of the normal samples wereobtained from non-smokers.

[0110] The smoke-exposed mouse is generally used as an animal model todevelop emphysema after 6 months of exposure to cigarette smoke.(35)Six-month-old mice were •subjected to smoke from two non-filteredcigarettes per day. After 6 months, the mice were sacrificed for lungexcision. A transgenic mouse model was used which overexpresses humaninterstitial collagenase in the lung develops emphysema.(7) The lungswere removed from five-month-old mice. For developmental analysis, thelungs of 14 and 18 dpc embryos and newborn wild-type mice werecollected. For induction of the acute phase reaction with LPS (Sigma,St. Louis, Mo.), mice were given intraperitoneal injections of saline orLPS. Animals were killed 24 h after intraperitoneal injections.(36)Total RNA was isolated from lung, heart, kidney and liver tissues asdescribed above.

[0111] Differential Display.

[0112] Total RNA was prepared from fresh tissue by the guanidiniumthiocyanate-cecium chloride method. Differential display was performedusing RNAimage (GeneHunter Corp., Nashville, Tenn.). Total RNA (0.2 mg)was reverse transcribed with three different one-base-anchored oligo(dT)primers (H-T, ,M, M=G, C or A). The reactions were performed for eachRNA sample in 25 mM Tris-HCl, pH 8.3, 37.6 mM KCl, 1.5 mM MgCl₂, 5 mMDTT, 20 uM of each dNTP, and 0.2 uM of H-T₁₁M. The solutions were heatedto 65° C. for 10 min and cooled at 37° C. for 10 min, after which 100 Uof MMLV reverse transcriptase were added. After incubation at 37° C. for50 min, the mixture was heated to 75° C. for 5 min to inactivate thetranscriptase before storage at −20° C. The PCR mixture contained 0.1volume of the RT reaction, 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 2.0 mMMgCl, 2 uM of each dGTP, dTTP and dGTP, a-[³³P]dATP (2,000 Ci/mmole,NEN, Boston, Mass.), 0.2 mM of arbitrary 13-mer primer, 0.2 mM of therespective H-T_(U)M oligonucleotide and Taq DNA polymerase (Gibco BRL,Gaithursberg, Md.). Each PCR amplification was carried out for 40 cyclesat 94° C. for 30 sec, at 40° C. for 2 min, and at 72° C. for 30 sec,followed by 5 min postextension at 72° C. Radiolabeled PCR amplificationproducts were analyzed by electrophoresis on denaturing 6%polyacrylamide gels, and the dried gels were exposed to X-ray film. Fourindependent samples from two emphysema lungs and two normal lungs werecompared side by side on gels to confirm the reproducibility of bandingpatterns.

[0113] Subcloning and Verification of Bands.

[0114] Bands of interest ranging from 150 to 800 bp were recovered fromthe gels and reamplified in a 40 cycle PCR reaction in the absence ofisotope. The reamplified PCR bands were subcloned into a pGEM-T EasyVector (Promega, Madison, Wis.). Individual clones (1 |ig) were spottedon a nitrocellulose membrane (Protran, Schleicher & Schuell, Keene,N.H.) in a 96-well format (Schleicher & Schuell). The membranes werethen hybridized with ³²P-labeled first strand cDNA prepared by RT oftotal RNA of normal or emphysema lungs using an oligo (dT) ₁₂₋₁₈ primer(Gibco BRL) . Hybridized clones to either the emphysema or normal lungprobe were used for sequence analysis. The sequences were queriedagainst the National Center for Biotechnology Information (NCBI)database using the Basic Local Alignment Search Tool (BLAST) algorism.To verify expression patterns of genes in vivo, DNA-free total RNA wasprepared and converted to a first-stranded cDNA using a random primer(Gibco BRL) and Superscript II (Gibco BRL) followed by PCRamplification. The primer sets (20-mer) to each gene were designed andused for verification of specific expression in either the normal oremphysema sample.

[0115] 5′RACE.

[0116] 5′RACE was performed basically according to the manufacturer'sinstruction (5′RACE System for Rapid Amplification of cDNA Ends, GibcoBRL). Poly(A) RNA was reverse transcribed with oligo(dT)₁₂₋₁₈ primer andSuperscript II followed by RNA digestion of the first strand cDNA. Ahomopolymeric tail was added to the 3′-end of the cDNA using terminaldeoxynucleotidyl transferase and dCTP, which allows hybridization to theAbridged Anchor Primer in subsequent PCR reactions. PCR reactions wereperformed with primer sets of the Abridged Anchor Primer and the genespecific primer, and Taq/Pwo DNA polymerase Mix (Boehringer Mannheim,Indianapolis, Ind.). The second PCR reaction was carried out using thefirst PCR reactions as templates, the Abridged Universal AmplificationPrimer and a 3′-nested gene specific primer. Amplified fragments weresubcloned into pGEM-T Easy Vector and sequenced. RT-PCR. Specific primersets used are shown below. sFRP1; Forward 5′-TACAAGAAGATGGTGCTGCC-3′,Reverse 5′-AGCACAAGCTTCTTCAGGTC-3′, Nested Reverse5′-AGATGTTCAATGATGGCCTC-3′: sFRP2; Forward 5′-TCTTCCTCTTTGGCCAGCCC-3′,Reverse 5′-TCACATCAATTTGGAGCTTC-3′: sFRP3; Forward5′-TCTGCACCATTGACTTCCAG-3′, Reverse 5′-TCTCAGCTATAGAGCCTTCC-3′, NestedReverse 5′-TTAGAATCTCCTTCACCTCC-3′: sFRP4- Forward5′-TCCTGGCCATCGAGCAGTAC-3′, Reverse 5′-GATGAGGACTTGAAGATCTC-3′: s F R P5; Forward 5′-ACTCGGATACGCAGGTCTTC-3′, Reverse5′-TTCTTGTCCCAGCGGTAGAC-3′: WNT1; Forward 5′-TCCTCCACGAACCTGCTTAC-3′,Reverse 5′-ACATCCCGTGGCACTTGCAC-3′, Nested Reverse5′-TTCGATGGAACCTTCTGAGC-3′: WNT5A; Forward 5′-GACAGAAGAAACTGTGCCAC-3′,Reverse 5′-TGTCTTCAGGCTACATGAGC-3′: W N T 8 B; Forward5′-CGCAAGTATCAGTTTGCCTG-3′, Reverse 5′-TAGAGATGGAGCGAAAGGTG-3′, NestedReverse 5′-TGGTACTTCTCCTTCAGGTG-3′: HZD2; Forward5′-TCTCAGCTACAAGTTTCTGG-3′, Reverse 5′-CCATGCTGAAGAAGTAGAGC-3′: HZD3;Forward 5′-TGTGCTACAACGTCTACTCG-3′, Reverse 5′-ATGAGCTTCTCCAGCTTCTC-3′:HFZ5; Forward 5′-TCCTATGCACTATGTACACG-3′, Reverse5′-TGTCCATGTCGATGAGGAAG-3′: HFZ6; Forward 5′-TGGATTTTGGTGTCCAAGGC-3′,Reverse 5′-AAGAATCACCCACCACACAG-3′: GAPDH; Forward5′-TTCCACCCATGGCAAATTCC-3′, Reverse 5′-TTTCTAGACGGCAGGTCAGG-3′: sFrp1;Forward 5′-AGCGACGTGCAAAAGGAGAG-3′, Reverse 5′-AGCCTGAAATGCCTCATGTC-3′:sFrp3\ Forward 5′-ACATGACCAAGATGCCCAAC-3′, Reverse5′-TCCCTTGGAATGTTTACCAG-3′: G a p d h; Forward5′-ATGCATCCTGTACCACCAAC-3′, Reverse 5′-TGGTCCTCTGTGTAAGCAAG-3′.

[0117] After the RT reaction of total RNA using Superscript II, the samePCR reaction described above was performed with an annealing temperatureof 52° C. for sFRP1 and 3, 58° C. for sFRP2, 4 and 5, WNT1, 5A and 8B,HZD2 and 3, HFZ5 and 6, and sFrp1 and 3, 50° C. for GAPDH and Gapdh.Nested PCR was carried out for sFRP1 and 3, and WNT1 and SB.(38)Amplicons were then analyzed on a 2% agarose gel.

EXAMPLE 3 Transfection of sFRP-1 Gene Induces Apoptosis WithoutPreference to Cell-Type Examined

[0118] We have shown that sFRP-1 is upregulated in emphysema throughdifferential display.

[0119] Then we demonstrated that sFRP-1 is a developmentally regulatedgene during lung development. Immunohistochemistry results show thatsFRP-1 is localized to the distal epithelial cells between day 13.5 and15.5 and then expression is turned off. This expression colocalizes tothe expression of wnt 10 b. Thus, wnt 1Ob appears to be inhibited bysFRP-1 during lung development and possibly also in emphysema.

[0120] SFRP-1 is hypothesized to play a role in apoptosis. In thisExample we demonstrate that apoptosis does occur in emphysema and mostrecently we have shown through transfection studies that increasedexpression of sFRP-1 leads to apoptosis in lung epithelial cells,endothelial cells and fibroblasts.

[0121] Methods

[0122] Histological Examination:

[0123] Immunohistochemical staining was performed using mouse IgG tohuman proliferating cell nuclear antigen (PCNA) (clone PC 10, 1 mg/ml,Sigma, St. Louis, Mo.) and goat IgG to human sFRP1 (clone sc7425, 4ug/ml, Santa Cruz Biotechnology, Santa Cruz, Calif.). For epitoperetrieval of sFRP1, tissue sections were processed to the microwavetreatment in 0.01 M sodium citrate buffer, pH 6.0 at 500 W. Biotinylatedhorse IgG to mouse IgG or FITC-labeled rabbit IgG to goat IgG were usedas secondary antibodies. An avidin-biotin-peroxidase complex coupledwith biotinylated hours IgG was visualized by 3,3′-diaminobenzidinetetrahydrochloride.

[0124] Apoptotic and Kproliferating Index:

[0125] Percentage of TUNEL- or PCNA-reactive cells (apoptotic index andproliferation index, respectively) was mesured among over 3,000 lungparenchymal cells in randomly serected areas in each specimens at40-fold magnification using light microscopy. The significance ofdifference in the apoptotic index or proliferation index among normaland four different clinical grade of emphysema was determined by one-wayanalysis of varience (ANOVA).

[0126] Protein Preparation and Analysis:

[0127] For immunological detection of sFRP1, Western blot was performedusing goat anti-sFRP1 antibody (1.5 mg/ml) and biotinylated rabbit IgGto goat IgG was used as a secondary antibody.

[0128] Transfection of Lung Cells with a sFrp1 Expression Vector:

[0129] The mouse sFrp1 full length cDNA was generously provided by Drs.Jeremy Nathans and Amir Rattner, John Hopkins University, Baltimore, andsubcloned into a pCMS-EGFP vector (Clontech, Palo Alto, Calif.). A geneof sFrp1w&s inserted into the multiple cloning site at EcoRI and Sailsites between the cytomegalovirus early promoter and SV40polyadenilation signals, which direct proper processing of the 3′ end ofsFrp1 mRNA. The vector also has a Gfp (green fluorescent protein) genelocated in downstream of the sFrp1-SV40 polyadenilation signals andligated between the SV40 enhancer/promoter sequence and apolyadenylation signal from the bovine growth hormone gene. This vectorconstruct allows transcriptions of sFrp1 and Gfp as separate proteins intransfected cells. Normal human primary cultured cells of small airwayepithelial cells (SAEC 6043), lung microvascular endothelial cells(HMVEC L 6521-3), and lung fibroblasts (NHLF 5975) were obtained fromBioWhittaker (Walkersville, Md.) and cultured in proper media containing10% fetal bovine serum. Cells were plated 2 days before transfection in4-well LabTek II chamber (Nalge Nunc International, Naperville, Ill.) at2×10⁴ cells/chamber and incubated at 37★C in 5% COj incubator. ThesFrp1-Gjp expression vector or Gfp vector without sFrp gene (0.8 ujj)were transfected into cells using Lipofectaniine PLUS according to themanufacture's instruction (Gibco BRL). For Annexin-V-Biotin assay (RocheDiagnostics GmbH, Manheim, Germany), ceils were harvested at 72, 48, 24,12 or 6 h after transfection and the reaction was detected byExtraAvidin-Cy3 (Sigma). For caspase 3 activation assay, cells werefixed in 3% paraformaldehyde in PBS after 24 or 48 h of transfection andincubated with anti-human active caspase 3 antibody made by rabbit(xlOO, clone 9661. Cell Signaling Technology, Beverly, Mass.).Biotinylated goat IgG to rabbit IgG (Vector) and ExtraAvidin-Cy3 (Sigma)were used for detection of binding of a primary antibody.

[0130] Results

[0131] Correlation of Morphometric Measurements with Apoptotic Index andProliferation Index.

[0132] Morphometric measurement showed progressive deterioration of lungarchitecture along with clinical grades of emphysema. The apoptoticindex was increased as clinical grades of emphysema going to progressedbut the index was decreased in the sever group. The statisticalsignificance was observed between normal-mild, normal-moderate, andnormal-sever. The mild grade also significantly higher of theprolifaration index than other grades. Through regression analysis theapoptotic index was shown to inversely correlate with the surface areademonstrating a close association of apoptosis and decrease of the !ungsurface area. Proliferation index did not have any correlation withapoptotic index and surface area. However, in normal lung, linearcorrelation was exhibited between proliferation index and apoptoticindex. This initial correlation suggests that overcome of apoptoticactivities to proliferation activities in emphysema lungs and viceversa. Apoptosis-activation by sFrp1 expression. Transfection studiesexhibited an apoptosis-inducing activity of sFrp1 without preference ofcell-type examined. The activation of translocation ofphosphatidylserine exposure to the outer cell membrane as detected byannexin V assay showed time- and dose-dependency of cell death to sFrp1expression (data not shown). Fluorescent microscope indicated sFrp1activity in autocine and paracrine fashion. Immunostaining analysisdemonstrated the presence of acive caspase 3 in and surrounding cellstransfected sFrp1, indicating apoptosis-activating activity of sFrp1.Further studies will be required to elucidate that anapoptosis-induction by sFrp1 activity is direct or indirect throughinhibition of endogenous Wnt.

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What is claimed is
 1. A method of treating or preventing a chronicobstructive pulmonary disease in a subject, comprising administering tosaid subject an amount of an agent effective to inhibit apoptosis of thesubject's lung cells and thus treat or prevent chronic obstructivepulmonary disease in the subject.
 2. The method of claim 1, wherein theagent inhibits the apoptosis by inhibiting expression of a secretedFrizzeled-related protein (sFRP) gene.
 3. The method of claim 2, whereinthe sFRP-1 gene encodes a polypeptide having the amino acid sequence setforth in SEQ ID NO:1.
 4. The method of claim 1, wherein the chronicobstructive pulmonary disease is emphysema.
 5. The method of claim 1,wherein the chronic obstructive pulmonary disease is chronic bronchitis.6. The method of claim 1, wherein the agent is selected from the groupconsisting of: an antisense molecule, a b-chemokine, and a plant-derivedcomposition.
 7. The method of claim 6, wherein the antisense moleculecomprises from eight (8) to thirty (30) nucleotides.
 8. A method ofidentifying a compound effective to treat or prevent a chronicobstructive pulmonary disease, comprising. a) contacting lung cells froma subject having a chronic obstructive pulmonary disease with thecompound and measuring the level of apoptosis of the lung cells in thepresence of said compound, b) measuring the level of apoptosis of lungcells from the same subject in the absence of said compound, and c)comparing the level of apoptosis in step a) with the level of apoptosisin step b), wherein a higher level of apoptosis in step b) indicatesthat the compound is effective to treat or prevent chronic obstructivepulmonary disease.
 9. The method of claim 8, wherein the level ofapoptosis is determined by measuring DNA fragmentation or cleavage. 10.The method of claim 8, wherein the level of apoptosis is determined bymeasuring the expression of activated caspase
 3. 11. The method of claim8, wherein the level of apoptosis is determined by measuring thepresence of poly(ADP ribose) polymerase.
 12. The method of claim 8,wherein the level of apoptosis is determined by morphometric analysis.13. The method of claim 8, wherein the level of apoptosis is determinedby measuring Bcl-2 and/or Bad expression.
 14. A method of treating orpreventing a chronic obstructive pulmonary disease in a subject,comprising administering to the subject an amount of an agent effectiveto inhibit expression of a secreted Frizzled-related protein (sFRP) geneof the subject's lung cells and thus treat or prevent chronicobstructive pulmonary disease in the subject.
 15. The method of claim14, wherein the chronic obstructive pulmonary disease is emphysema orchronic bronchitis.
 16. An antibody capable of specifically binding tosFRP-1.
 17. The antibody of claim 16, wherein the antibody is amonoclonal antibody.
 18. The antibody of claim 16, wherein the antibodyis a polyclonal antibody.
 19. The antibody of claim 16, wherein theantibody is humanized.
 20. The antibody of claim 16, wherein theantibody is detectable.
 21. The antibody of claim 20, wherein thedetectable antibody is labeled with a detectable marker.
 22. The labeledantibody of claim 21, wherein the detectable marker is a radioactivelabel or a calorimetric, or a luminescent, or a fluorescent marker. 23.A composition comprising the antibody of claim 16 and an agentconjugated to the antibody.
 24. The composition of claim 23 wherein theagent is a radioactive isotope or toxin.
 25. A method of determiningwhether a subject is afflicted with a chronic obstructive pulmonarydisease which comprises: (a) obtaining a suitable sample from thesubject; (b) contacting the suitable sample with the detectable antibodyof claim 16 so as to form a complex between the antibody and sFRP orfragment thereof present in the sample; (c) removing any unboundantibody; and (d) detecting any antibody which is bound to any sFRP inthe sample, wherein the presence of antibody indicates that the subjectis afflicted with the chronic obstructive pulmonary disease.
 26. Themethod of claim 25, wherein the disease is emphysema.
 27. The method ofany one of claim 25, wherein the suitable sample is lung tissue.
 28. Themethod of claim 25, wherein the antigen bound by the antibody isdetected by an immunoassay.
 29. The method of claim 25, wherein theimmunoassay is ELISA.
 30. The method of claim 25, wherein theimmunoassay is IFA.
 31. The method of claim 25, wherein the immunoassayis Western blotting.
 32. A kit for diagnosing chronic obstructivepulmonary disease comprising the labeled antibody of claim
 21. 33. Thekit of claim 32, further comprising a means for determining the level ofsFRP or fragment thereof bound by an antibody.
 34. The kit of claim 32,wherein the antibody is bound to a support.
 35. A method of inhibitingsFRP mediated apoptosis of a cell which comprises introducing into thecell an effective amount of the replicable vector which expresses anantisense molecule to the gene encoding sFRP so as to thereby inhibitsFRP mediated apoptosis of the cell.
 36. The method of claim 35, whereinthe sFRP is sFRP-1.
 37. A method for evaluating in a non-humantransgenic animal the potential therapeutic effect of an agent fortreating chronic obstructive pulmonary disease in a human, whichcomprises: (a) providing an agent to a transgenic non-human animalhaving chronic obstructive pulmonary disease; (b) determining thetherapeutic effect of the agent on the transgenic non-human animal bymonitoring sFRP expression, wherein a decrease in sFRP indicates thatthe agent would have a potential therapeutic effect on chronicobstructive pulmonary disease in a human.
 38. The method of claim 37,wherein the animal is a mammal.
 39. The method of claim 37, wherein thenon-human animal is a mouse, a rat, a sheep, a dog, a primate, or areptile.
 40. A method of detecting a chronic obstructive pulmonarydisease in a subject which comprises: a) obtaining a suitable sample ofmRNA from the subject; b) contacting the mRNA sample under hybridizingconditions with a labeled nucleic acid probe which: (1) is at least 15nucleotides in length and (2) hybridizes specifically to a nucleic acidhaving a sequence which is complementary to a sequence present in thesequence set forth in SEQ ID NO. 2; c) removing any unbound labelednucleic acid probe; and d) detecting the presence of labeled nucleicacid probe hybridized to the mRNA so as to thereby detect chronicobstructive pulmonary disease in the subject.
 41. The method of claim40, wherein the mRNA is form lung tissue of the subject.
 42. A method ofdetecting chronic obstructive pulmonary disease in a subject whichcomprises: a) obtaining a suitable sample of mRNA from the subject; b)reverse transcribing the mRNA to generate a single-stranded cDNA; c)contacting the single-stranded cDNA under hybridizing conditions with alabeled nucleic acid probe which: 1) is at least 15 nucleotides inlength; and 2) hybridizes specifically to a nucleic acid having asequence set forth in SEQ ID NO:2; d) removing any unbound labelednucleic acid probe; and e) detecting the presence of labeled nucleicacid probe hybridized to the cDNA so as to thereby detect detect chronicobstructive pulmonary disease in the subject.
 43. A method of detectingchronic obstructive pulmonary disease in a subject which comprises: a)obtaining a suitable sample of mRNA from the subject; b) generating adouble-stranded mRNA-cDNA duplex from the mRNA; c) contacting the duplexfrom (b) with one primer having a sequence which is complementary to aportion of the sequence set forth in SEQ ID NO:2 and a second primerhaving a sequence which comprises a different portion of the sequenceset forth in SEQ ID NO:2; d) amplifying the nucleic acid from (c) usinga polymerase chain reaction to obtain an amplification product; e)contacting the amplification product of (d) under hybridizing conditionswith a labeled nucleic acid probe which: 1) is at least 15 nucleotidesin length; 2) hybridizes specifically to a nucleic acid having asequence set forth in SEQ ID NO. 2; f) removing any unbound labelednucleic acid probe; and g) detecting the presence of labeled nucleicacid probe hybridized to the amplification product so as to therebydetect chronic obstructive pulmonary disease in the subject.